The isolation of nucleic acids such as, for example, DNA or RNA is an important step in many biochemical and diagnostic procedures. For example, the separation of nucleic acids from the complex mixtures in which they are often found is frequently necessary before other studies and procedures e.g. detection, cloning, sequencing, amplification, hybridisation, cDNA synthesis, studying nucleic acid structure and composition (e.g. the methylation pattern of DNA) etc can be undertaken. The presence of large amounts of cellular or other contaminating material e.g. proteins or carbohydrates, in such complex mixtures often impedes many of the reactions and techniques used in molecular biology. In addition, DNA may contaminate RNA preparations and vice versa. Thus, methods for the isolation of nucleic acids from complex mixtures such as cells, tissues etc. are demanded, not only from the preparative point of view, but also in the many methods in use today which rely on the identification of DNA or RNA e.g. diagnosis of microbial infections, forensic science, tissue and blood typing, genotyping, detection of genetic variations etc. The purification of DNA or RNA from more enriched but still contaminated samples is also desirable, e.g. to purify synthetically prepared nucleic acid material, e.g. to purify PCR products from contaminating salts, excess primers and/or dNTPs.
A range of methods are known for the isolation of nucleic acids, but generally speaking, these rely on a complex series of extraction and washing steps and are time consuming and labourious to perform. Moreover, the use of materials such as alcohols and other organic solvents, chaotropes and proteinases is often involved, which is disadvantageous since such materials tend to interfere with many enzymic reactions and other downstream processing applications.
Thus, classical methods for the isolation of nucleic acids from complex starting materials such as blood or blood products or tissues involve lysis of the biological material by a detergent or chaotrope, possibly in the presence of a protein degrading enzyme, followed by several extractions with organic solvents e.g. phenol and/or chloroform, ethanol precipitation, centrifugations and dialysis of the nucleic acids. The purification of RNA from DNA may involve a selective precipitation with LiCl or a selective isolation with acidic guanidinium thiocyanate combined with phenol extractions and ethanol precipitation. Not only are such methods cumbersome and time consuming to perform, but the relatively large number of steps required increases the risk of degradation, sample loss or cross-contamination of samples where several samples are simultaneously processed.
One approach common in RNA purification, which may be used in conjunction with the solid phase approach is to carry out the lysis of the biological material and the subsequent hybridisation to oligodT in LiCl and LiDS/SDS buffers, thereby avoiding extra steps such as phenol extraction or proteinase-K digestion. The whole direct mRNA isolation takes approximately 15 minutes and since the mRNA is stable for more than 30 minutes in the lysis buffer, this ensures the high quality of the mRNA purified. However, a disadvantage of this method is that mRNA per weight unit of tissue is affected by the amount of tissue used and above a critical threshold of lysed cells, the yield of mRNA decreases.
Another common approach for direct mRNA purification is, to use guanidinium isothiocyanate (GTC) and sarkosyl. A GTC-buffer system is preferred by most researchers due to the ability of this chaotropic salt to inhibit RNases. This may also be used in combination with the magnetic bead approach.
However, the viscosity of cell lysates in 4M GTC is high and the beads are not effectively attracted by the magnet, resulting in an increased risk for DNA contamination, both for beads and other solid phases, and lower yields.
More recently, other methods have been proposed which rely upon the use of a solid phase. U.S. Pat. No. 5,234,809, for example, describes a method where nucleic acids are bound to a solid phase in the form of silica particles, in the presence of a chaotropic agent such as a guanidinium salt, and thereby separated from the remainder of the sample. WO 91/12079 describes a method whereby nucleic acid is trapped on the surface of a solid phase by precipitation. Generally speaking, monohydric alcohols (ethanol or isopropanol) and salts are used as precipitants.
Ethanol and/or isopropanol when included in sufficient quantity in an aqueous medium act to precipitate dissolved nucleic acids out of solution and to keep out of solution nucleic acids already in the extra-solution phase. Ethanol and isopropanol may therefore be described as “anti-solvating” agents for nucleic acids. Without being bound by theory, it is believed that the ethanol or isopropanol reduce the amount of free water available to dissolve the nucleic acid. These two monohydric alcohols are therefore used during the processing of nucleic acids to precipitate them (e.g. bind them to a solid support) and/or to keep them from dissolving in the aqueous medium, in the latter case while other procedures are carried out. In any particular workflow, one or a combination of these alcohols may be used to precipitate nucleic acid, to keep nucleic acid precipitated, or both to precipitate nucleic acid and to keep it precipitated.
U.S. Pat. No. 5,705,628 and U.S. Pat. No. 5,898,071 describe methods of isolating nucleic acid fragments using a combination of large molecular weight polyalkylene glycols (e.g. polyethylene glycols) at concentrations of from 7 to 13% with salt in the range of 0.5 to 5M to achieve binding to functional groups on a solid support which acts as a bioaffinity absorbent for DNA.
Although such methods generally speed up the nucleic acid separation process, there are disadvantages associated with basing procedures involving solid phase nucleic acids on current agents for inducing and/or maintaining the precipitated state, for example alcohols (such as ethanol or isopropanol), chaotropes, salts and large molecular weight molecules.
Large molecular weight molecules increase the viscosity of the liquid which reduces the efficiency with which purification protocols can be conducted. In the case of separation of magnetic beads, such large molecules reduce the speed of isolation as the time of contact with the magnet to separate the beads has to be increased. Furthermore, the removal of supernatant in such systems is more difficult in the presence of the large molecular weight molecules.
Chaotropes need to be used at high molarity, resulting in viscous solutions which may be difficult to work with, especially in RNA work. Amplification procedures such as PCR, and other enzyme-based reactions, are very sensitive to the inhibitory or otherwise interfering effects of alcohols and other agents. Moreover, the drying of the nucleic acid pellet which is necessary following alcohol (i.e. ethanol or isopropanol) precipitation and the problems with dissolving nucleic acids, are also known to lead to artefacts in enzyme-based procedures such as PCR. Furthermore, liquids with a high content of ethanol or isopropanol are volatile and flammable, which increases transport and disposal costs for the liquids and hence increases the cost of the overall process.
Since such preparation procedures are now a mainstay of molecular biology, there is a need for improved methods of nucleic acid isolation, and particularly for methods which are quick and simple to perform, which enable good yields to be obtained with minimal losses, and which avoid the use of volatile solvents and chaotropic agents or alcohol precipitation or the use of high levels of salt and/or high molecular weight compounds with high viscosity.
A replacement for alcohol in these known preparation procedures would advantageously function as anti-solvating agent at a final concentration that does not increase the volume of the system in order to work in established automated assays.
It would be desirable to be able to use another agent than those currently used to remove nucleic acids from solution or to keep them out of solution whilst contacting them with a liquid medium. It would be more advantageous to be able to remove nucleic acids from solution, or to keep them out of solution whilst contacting them with a liquid medium, by using an agent which did not involve to the same extent the shortcomings of the agents currently used for these purposes.